The clinical samples of lung cancer tissue and paired normal adjacent tissues were collected from lung cancer patients at Zhuji Affiliated Hospital of Shaoxing University in 2019 for treatment. A total of 72 patients with lung cancer (age, 35–70 years) were enrolled between December 2018 and December 2019. The male to female ratio was 3:1. Patients were divided into three groups according to the age of onset as follows: Group 1, 35–50; group 2, 50–62; group 3, 62–70. Cancer and normal adjacent tissues of all patients were obtained and used to detect the gene expression level of BRF2 in the tissues. The patients had no history of chemotherapy and did not have other types of cancer, infectious diseases, or autoimmune diseases. All patients signed informed consent and agreed that their tissues would be used for clinical research. The study was approved by the Ethics Committee of Zhuji Affiliated Hospital of Shaoxing University (approval no. ZLK20181124). All clinical samples were obtained at the time of initial resection, and stored at −80°C.

BRF2 mRNA expression levels in lung cancer and normal tissues were compared using a bioinformatics website (gepia.cancer-pku.cn).

Normal human lung epithelial cells (BEAS-2B; cat. no. CRL-9609) and human lung cancer cells (A549, cat. no. CCL-185; H292, cat. no. CRL-1848) were purchased from the American Type Culture Collection. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich; Merck KGaA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO₂.

The cells (2×10⁴/ml) were grown on coverslips and after the liquid had been aspirated, the cells were covered 2–3 mm under 4% formaldehyde diluted in warm phosphate-buffered saline (PBS) to be fixed for 15 min at room temperature. Following removal of the fixative, the cells were rinsed in PBS with for 5 min. Then the cells were blocked with immunostaining blocking buffer (cat. no. P0102; Beyotime Institute of Biotechnology) at 37°C for 60 min, after aspirating the solution, primary antibody anti-BRF2 antibody (mouse, cat. no. ab194442, 1:500, Abcam) was added to the cells. Next, the cells were incubated overnight at 4°C. The cells were rinsed 3 times in PBS for 5 min at room temperature in the dark, and then incubated with fluorochrome-conjugated secondary antibody [horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG H&L (1:100; cat. no. ab6789; Abcam)] at 37°C for 1 h. DAPI (cat. no. D1306, Thermo Fisher Scientific, Inc.) was added to the cells and incubated together for 5 min in the dark. Following aspiration of the liquid, the cells were observed under a fluorescence microscope (magnification, ×200; cat. no. BX43; Olympus Corporation).

Following fixation in 4% formaldehyde at 25°C for 24 h, the tissues were dehydrated and made transparent by gradient alcohol, then paraffin-embedded, and sectioned (section thickness, 5 µm). The sections were soaked in citrate buffer solution (pH 6.0) and heated in a 850 W power microwave oven for 10 min to conduct antigen repair. The endogenous peroxidase enzyme activity was minimized by rinsing the sections in 3% H₂O₂ in methanol for 20 min at room temperature. Then, the tissues were incubated with primary rabbit anti-BRF2 polyclonal antibody (1:400, cat. no. ab194442, Abcam) at 4°C overnight. Next the sections were incubated with secondary antibody [HRP-conjugated goat anti-rabbit immunoglobulin (IgG) H&L (1:2,000; cat. no. ab150113; Abcam) and streptavidin-peroxidase complex for 30 min at 37°C. The sections were stained with hematoxylin, dried in an oven at 65°C, rinsed in water, then mixed with alcohol and xylene and naturally dried. Finally, the sections were sealed and observed under an optical microscope (magnification, ×100; BX53M, Olympus Corporation).

BRF2 small interfering (si)RNA (cat. no. 11968S, Cell Signaling Technology, Inc.) was transfected into the A549 cells using Lipofectamine^® 2000 Transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A549 cells were seeded into 6-well plates at 1×10⁵ cells per well and incubated at 37°C for 24 h. Then 1.5 ml medium without serum or antibiotics was added into each well of the plate, with the mixture of 100 pmol BRF2 siRNA and Lipofectamine^® 2000 to incubate for 4–6 h at 37°C with 5% CO₂. The siRNA sequences were as follows: BRF2 siRNA sense, 5′-GGUGGGAAAUAAUUCCUUATT-3′; si-negative control (siNC) sense, 5′-UUCUCCGAACGUGUCACGUTT-3′. After 72 h of transfection, the cells were collected for later analysis.

Total RNAs were extracted from tissues or cells (2×10⁴/ml) by TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Total RNAs were placed in a refrigerator at 4°C, and quantified by a biological spectrometer (Nano Drop 2000C; Thermo Fisher Scientific, Inc.). The extracted RNAs were reverse-transcribed to cDNA using a Prime Script RT reagent kit (Takara Bio, Inc.) according to the manufacturer's instructions. The results were normalized to β-actin. Next, SYBR^® Green PCR Master Mix (cat. no. 4312704, Applied Biosystems, Thermo Fisher Scientific, Inc.) and Bio-Rad CFX 96 Touch Real-Time PCR Detection system (cat. no. 1855196, Bio-Rad Laboratories, Inc.) were used for RT-qPCR, according to the manufacturer's instructions. The thermocycling conditions were as follows: 95°C for 5 min, 40 cycles of 95°C for 15 sec, 60°C for 30 sec and 70°C for 10 sec. The primers for β-actin and BRF2 were: β-actin forward, 5′-ATTGGCAATGAGCGGTTC-3′ and reverse: 5′-GGATGCCACAGGACTCCA-3′; BRF2 forward, 5′-CACAGGGGAAAACGAACAAG-3′ and reverse, 5′-TCGACAAAGGTCTCTCACTCG-3′. The gene expression was calculated by the 2^−ΔΔCq method (21). Each experiment was performed 3 times.

Following transfection for 48 h, A549 cells were collected for western blot analysis as previously described (22). Total proteins were extracted from the cells by RIPA lysis buffer (Thermo Fisher Scientific, Inc.), and protein concentration was determined by bicinchoninic protein assay kit (Sigma-Aldrich; Merck KGaA). The proteins (10 µg) were separated by 10% SDS-PAGE (cat. no. P0012A, Beyotime Institute of Biotechnology) and transferred onto polyvinylidene fluoride membranes (cat. no. FFP28, Beyotime Institute of Biotechnology), which were blocked with 5% fat-free milk for 1 h at room temperature. The membranes were then incubated overnight at 4°C with the primary antibodies: anti-Akt antibody (rabbit, cat. no. ab8805, 1:500, Abcam), anti-p-Akt antibody (rabbit, cat. no. ab38449, 1:500, Abcam), anti-Bax antibody (rabbit, cat. no. ab32503, 1:1,000, Abcam), anti-Bcl-2 antibody (rabbit, cat. no. ab59348, 1:500, Abcam), anti- E-cadherin antibody (rabbit, cat. no. ab40772, 1:1,000, Abcam), anti-N-cadherin (rabbit, cat. no. ab18203, 1 µg/ml, Abcam), anti-Snail antibody (rabbit, cat. no. ab229701, 1:1,000, Abcam), anti-EGFR antibody (rabbit, cat. no. ab52849, 1:1,000, Abcam) and anti-β-actin antibody (mouse, cat. no. ab8226, 1:500, Abcam). β-actin served as an internal reference. The membranes were then incubated with secondary HRP-conjugated antibodies [goat anti-rabbit IgG H&L (HRP) (1:2,000; cat. no. ab150113; Abcam) and goat anti-mouse IgG H&L (HRP) (1:2,000; cat. no. ab6789; Abcam] at 37°C for 1 h and washed 3 times with 0.9% TBST. The protein bands were developed by an enhanced chemiluminescence (ECL) kit (Millipore), and the grey values of the strips were calculated by ImageJ (version 5.0; National Institutes of Health) (23).

The A549 cells were incubated in 96-well plates at a density of 5×10³ cells/well. Following transfection, the cells were cultured for 48 h with 5% CO₂ at 37°C. Next, 20 µl MTT (Promega Corp.) was added to each well. The formazan products were dissolved in 100 µl dimethylsulfoxide (DMSO), and the absorbance was detected at 540 nm using a microplate reader (PR3100 TSC, Bio-Rad Laboratories, Inc.). The cells were subjected to a multifunctional enzyme-linked analyzer (Attune NxT; Thermo Fisher Scientific, Inc.) for 4 h, and the absorbance value of each hole was measured at 490 nm.

The cells (5×10³ cells/well) from the different treatment groups were seeded in a 96-well plate, and cultured in an incubator with 5% CO₂ at 37°C for 48 h. Next, 100 µl CCK-8 and serum-free basic medium (DMEM; Thermo Fisher Scientific, Inc.) were mixed at a 1:10 ratio and added to a cell culture plate and cultured for 4 h. Finally, the absorbance at 450 nm wavelength was determined using a microplate reader (RNE90002, Reagen Biology LLC).

Following transfection, the A549 cells were washed with cold PBS twice. Annexin V/Dead Cell Apoptosis kit (cat. no. V13242, Thermo Fisher Scientific, Inc.) was used to identify apoptotic cells. Briefly, the cells were re-suspended in Annexin V binding buffer, added with Annexin V-FITC and propidium iodide (PI) buffer, and incubated at room temperature for 15 min in the dark. Cell apoptosis was analyzed using a flow cytometer (FACSCalibur™; version 2.0; BD Biosciences).

Following transfection, the cells were subcultured in plates at a density of 1×10⁵ cells/well and incubated in serum without FBS at 37°C for 24 h. The wounds were created using a 200-µl yellow sterile pipette tip on the monolayer, and detached cells were removed by washing the cells 3 times. The healing process was observed under a 600 Autobiochemical Analyzer (Olympus Corporation), and ImageJ software (Image Pro Plus; version 6.0; National Institutes of Health) was used to calculate the average distance between cells.

Matrigel (BD Biosciences) diluted at a 1:8 ratio was used to cover the upper surface of the membrane of the bottom of Transwell chambers, and the chambers were incubated at 37°C for 30 min in order to polymerize the Matrigel into gel. The cells were resuspended in the upper chambers of the Transwell (8-µm pore size, Corning Inc.), which contained serum-free DMEM medium, while the lower chambers were supplemented with DMEM containing 10% FBS. The Transwell chambers were all incubated for 48 h. Next, the cells remaining in the upper chamber were gently removed using a cotton swab, while the invaded cells were fixed in 4% paraformaldehyde for 15 min and stained by 0.1% crystal violet staining at 37°C for 20 min. The cells were randomly counted under an inverted microscope (Eclipse Ti2, Nikon Corporation) and images were captured.

All data were statistically analyzed by SPSS version 18.0 (SPSS, Inc.). The data are presented as means ± standard deviation, and all experiments were repeated 3 times. Two-group comparisons were conducted by the Student's t-test, and multi-group comparisons were conducted by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether BRF2 is involved in tumor migration and invasion, its protein expression in normal human lung epithelial BEAS-2B cells and lung cancer cells A549 and H292 were detected by immunofluorescence staining. As shown in Fig. 1A, marker protein BRF2 was positively expressed in A549 and H292 cells, especially in the A549 cells, while in BEAS-2B cells, marker protein BRF2 demonstrated a low expression. Meanwhile, BRF2 expression was detected by western blotting, as shown in Fig. 1B and C; BRF2 expression was significantly increased in A549 and H292 cells compared to that noted in the BEAS-2B cells. In addition, according to Fig. 1D, BRF2 protein staining was detected in cancer tissues by immunohistochemistry; however, BRF2 was barely observed in normal lung tissues. As shown in Fig. 1E, the BRF2 mRNA expression levels in lung cancer tissues and normal tissues were compared using a bioinformatics website (gepia.cancer-pku.cn), and the result demonstrated that the BRF2 mRNA expression level in the tumor tissues was notably higher compared with normal tissues. The BRF2 mRNA expression in lung cancer A549 and H292 cells and normal lung epithelial BEAS-2B cells was detected by RT-qPCR. Relative BRF2 mRNA expression level was the highest in A549 cells (Fig. 1F, P<0.001) and its protein expression was also high in H292 cells, while the mRNA expression level of BRF2 in BEAS-2B cells was low. In addition, normal adjacent and cancer tissues were divided into groups 1–3. Relative BRF2 mRNA expression level in lung cancer cells was clearly higher than normal cells (Fig. 1G, P<0.001).

To explore the role of BRF2 in lung cancer cells, A549 cells were transfected with siRNA for silencing BRF2. As shown in Fig. 2A, 48 h after BRF2 siRNA transfection, the result of RT-qPCR demonstrated that the relative mRNA expression level of BRF2 in A549 cells was significantly lower than that in the siNC group (P<0.001). The result of western blotting (Fig. 2B) demonstrated that the relative protein expression level of BRF2 was significantly inhibited (P<0.001 vs. siNC). In addition, the results of the CCK-8 and MTT assays demonstrated that after transfection for 48 h the relative cell viability of A549 cells in the siRNA group was significantly inhibited compared with the siNC group (Fig. 2C and D, **P<0.01).

Following transfection, the effects of siRNA on cell apoptosis and migration were assessed. As shown in Fig. 3A, flow cytometry demonstrated that the rate of apoptosis of the siRNA group was significantly higher compared with that noted in the siNC group (Fig. 3A, P<0.001). Meanwhile, cell migration and invasion were detected by wound-healing and Transwell assays. As shown in Fig. 3B, it was observed that the relative migration rate of the siRNA group was significantly lower than that noted in the siNC group (Fig. 3B, P<0.01). Furthermore, the result of Fig. 3C revealed that invasion of A549 cells transfected with BRF2 siRNA was significantly reduced (Fig. 3C, P<0.001).

The western blot analysis (Fig. 4A and B) revealed that following transfection of BRF2 siRNA, relative protein expression levels of Akt (P<0.01 vs. siNC), p-Akt (P<0.001 vs. siNC) and Bcl-2 (P<0.01 vs. siNC) were suppressed, while relative protein expression level of Bax was significantly promoted. Furthermore, as demonstrated in Fig. 4C, relative protein expression levels of N-cadherin (P<0.01 vs. siNC), Snail (P<0.01 vs. siNC) and EGFR (P<0.001 vs. siNC) were significantly inhibited. Notably, protein expression of E-cadherin (P<0.01 vs. siNC) was significantly promoted following transfection of BRF2 siRNA. In addition, the results further confirmed that BRF2 siRNA suppressed phosphorylation of Akt (Fig. 4D, P<0.001 vs. siNC).